The Role of Methylation in Chronic Lymphocytic Leukemia and Its Prognostic and Therapeutic Impacts in the Disease: A Systematic Review

Epigenetic regulation has been thoroughly investigated in recent years and has emerged as an important aspect of chronic lymphocytic leukemia (CLL) biology. Characteristic aberrant features such as methylation patterns and global DNA hypomethylation were the early findings of the research during the last decades. The investigation in this field led to the identification of a large number of genes where methylation features correlated with important clinical and laboratory parameters. Gene-specific analyses investigated methylation in the gene body enhancer regions as well as promoter regions. The findings included genes and proteins involved in key pathways that play central roles in the pathophysiology of the disease. Τhe application of these findings beyond the theoretical understanding can not only lead to the creation of prognostic and predictive models and scores but also to the design of novel therapeutic agents. The following is a review focusing on the present knowledge about single gene/gene promoter methylation or mRNA expression in CLL cases as well as records of older data that have been published in past papers.


Introduction
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal B cells in peripheral blood, bone marrow, and secondary lymphoid organs.It is the most common leukemia in adults of Western countries and accounts for approximately 2-35 percent of all leukemias in the United States [1].CLL is more common in men with a male to female ratio ranging from 1.2 :1 to 1.8 :1 [1,2] and is considered to be a disease of the elderly as the median age at diagnosis is 70 years and an incidence increasing rapidly with increasing age [3].Despite characterized by a common morphology and immunophenotype pattern, CLL shows a variable clinical behavior spectrum.At one end, some CLL patients have a very indolent disease course and may not require treatment for many years, while at the other end, some patients present a very aggressive disease early from diagnosis and require prompt treatment with lower survival rates despite therapy [4].
In terms of genetic pathobiology, at least one of the four common chromosomal abnormalities (deletion 13q14, trisomy 12, deletion 11q22-23, and deletion 17p12) can be detected by interphase fuorescence in situ hybridization in most patients [5].Most CLL tumors have myriads of somatic gene mutations.Of these, tumor protein 53 (TP53), ataxia telangiectasia mutated (ATM), neurogenic locus, notch homolog protein (NOTCH), and subunit 1 of the splicing factor 3b protein complex (SF3B1) genes are the most frequently mutated at diagnosis [6,7].Several other gene mutations seen in CLL tumors are involved in important cellular signaling pathways.NOTCH signaling, Bcell receptor (BCR) signaling, Toll-like receptor, mitogenactivated protein kinase/extracellular signal-regulated kinase (MAPK-Erk) pathway, nuclear factor kappa-lightchain enhancer of activated B cells (NF-kB) signaling, chromatin modifers signaling, cell cycle signaling, DNA damage signaling, and RNA splicing are some of the many pathways investigated [6][7][8][9][10].Moreover, it is well documented that the interaction between CLL tumor cells and antigens is regulated by the somatic hypermutation load of the immunoglobulin heavy chain variable region genes [11].
In the complex domain of the tumor microenvironment, CLL cells seem to infuence the kind and arrangement of the cells surrounding them, and they are highly dependent on signals from these cells for their own survival and proliferation, encouraging a leukemia-supportive and immunosuppressive microenvironment [11].
Methylation of DNA is an enzyme-mediated modifcation of DNA structure without interfering in the specifc sequence of the base pairs for the gene encoded.Although DNA methylation is important in normal biologic processes, aberrant patterns of methylation are observed in several malignancies.Specifcally, two patterns have been mainly described, namely, large sites of global hypomethylation along the genome and localized areas of hypermethylation at CpG islands, within the gene promoter regions.Ιn more and more studies in neoplastic diseases in recent years, there has been an emergence of evidence that methylation of the promoter regions of hundreds of genes, including tumor suppressor genes, results in the failure to express their normal purpose.In other cases, DNA methylation may represent an early step in the pathway by which normal tissue cells undergo tumorigenesis.Some other possible mechanisms for mutational induction secondary to DNA methylation include failure to produce DNA repair proteins that normally protect from mutations and predisposition to increased oxidative DNA damage, resulting in increased mutation potential [12].
Abnormal DNA methylation has been documented in many solid neoplasms, including breast cancer, lung, prostate, colorectal cancer, and melanoma.As described above, hypermethylation of promoter regions inhibits the expression of tumor suppressor genes while hypomethylation activates an oncogene expression.Global hypomethylation is also of crucial role, leading to genomic and chromosomal instability.For instance, BRCA1/2 genes are two of the most studied genes in breast cancer.Te hypermethylation of their promoter regions is reported to result in their inactivation and consequent increased risk of breast cancer [13].
As regards CLL, a global DNA methylation pattern was reported to be relatively stable during the disease course and similar both in resting and proliferative cell compartments, implying that aberrant methylation may present as an early leukemogenic event with early indications of specifc gene epigenetic changes in CLL samples being described over 30 years ago [14].Global DNA hypomethylation was reported few years later [15].
Except CLL, abnormal function of basic methylationrelated enzymes is widely reported in several hematological malignancies, such as in myelodysplastic syndromes, myeloproliferative neoplasms, acute myeloid leukemia, T-cell acute lymphoblastic leukemia, and difuse large B-cell lymphoma.Te fnal DNA methylation patterns are suggested to be frequently lineage specifc and accompanied by specifc secondary mutations [16].
Te research in this feld led to the study and discovery of a large number of genes whose methylation plays an important role in their regulation, while certain correlations with clinical and laboratory parameters were also identifed.Te streptothricin acetyltransferase, alpha (SAT-α) gene, whose methylation levels were found to be an independent marker of poor treatment-free survival was an early example [17].
With the appearance and application of newer technics such as whole-genome bisulfte sequencing and DNA methylation arrays, global DNA hypomethylation was confrmed and it was reported that gene body and enhancer regions rather than promoter regions were primarily involved [18,19] whereas hypermethylation was described to occur mainly in transcribed genomic regions [20] and regions afecting other regulatory mechanisms such as spliceosome [21].After the CLL genome was discovered to be globally hypomethylated, research for aberrantly methylated oncogene targets revealed that hypomethylation of the B cell lymphoma 2 (BCL2) gene, an important antiapoptotic gene, correlated with higher protein expression in CLL [22].In the following studies, multiple drug-resistance protein 1 (MDR1) and T-cell leukemia/lymphoma 1 (TCL1) genes were reported to be both hypomethylated and upregulated in CLL [23,24].Moreover, the nuclear factor of activated T-cell 1 (NFATc1) gene hypomethylation was also identifed and was further shown to be associated with increased mRNA and protein expression, suggesting hypomethylation as a mechanism of constitutive activation of NFATc1 expression in CLL.Trough subsequent fndings, however, it was discovered that the activation of oncogenes through DNA hypomethylation was a rather infrequent lesion in CLL [25].
To date, several studies have reported gene promoter hypermethylation in CLL patients using both global and single gene approaches.Te afected genes include tumor suppressors, transcription factors, genes involved in survival and proliferation, genes with prognostic impact, and microRNA genes.Tere are several methods that have been used to study gene methylation in chronic lymphocytic 2 Advances in Hematology leukemia.Each approach has its own advantages and limitations, and the choice of the method depends on the specifc research question being addressed.Bisulfte sequencing is considered the gold standard method for analyzing DNA methylation at single-nucleotide resolution.Bisulfte treatment of DNA converts unmethylated cytosine to uracil, while leaving methylated cytosine unchanged.
After polymerisation chain reaction (PCR) amplifcation and sequencing, the pattern of cytosine-to-thymine conversion can be used to determine the methylation status of individual CpG sites.Methylation-specifc PCR is a PCRbased method that uses primers specifc for either methylated or unmethylated DNA to amplify a region of interest.By comparing the intensity of PCR products amplifed using methylated-specifc versus unmethylated-specifc primers, the methylation status of a given region can be determined.Infnium methylation arrays are microarray-based platforms that can interrogate the DNA methylation status of thousands of CpG sites across the genome.Infnium arrays rely on bisulfte conversion of DNA, followed by hybridization with probes that distinguish between methylated and unmethylated DNA at each CpG site.Methylation-sensitive restriction enzyme digestion relies on the use of restriction enzymes that are sensitive to DNA methylation.By digesting genomic DNA with these enzymes and comparing the resulting fragment patterns with and without prior treatment with a DNA methyltransferase, the methylation status of specifc regions can be inferred.
When DNA methylation was studied in paired diagnostic and follow-up samples from IGVH mutated and unmutated CLL patients, genes with prognostic signifcance including chronic lymphocytic leukemia upregulated 1 (CLLU1), lipoprotein lipase (LPL), zeta chain of T-cell receptor-associated protein kinase 70 (ZAP70) and NOTCH1, the epigenetic regulators histone deacetylases 9 and 4 (HDAC9/4), and DNA methyltransferase 3, beta (DNMT3B), were reported to be aberrantly methylated [14].Another study used the methylation levels of fve specifc CpGs to track the cellular origin of CLL, where they seemed to act as enhancers.Te varying methylation levels of these biomarkers did not translate into gene expression changes, suggesting that they may not have a functional impact but represented a stable molecular mark [28].
In the context of normal B-cell maturation, CLL DNA methylation was reported to be highly enriched in enhancer and promoter regions, especially in regions of transcriptional elongation and in genes involved in B cell-and lymphocyte-related processes and pathways.Regions targeted for hypomethylation during B cell maturation showed highly signifcant enrichment for the following six transcription factor families: activator protein 1 (AP-1), early Bcell factor (EBF), runt-related transcription factor (RUNX), octamer-binding transcription factor (OCT), interferon regulatory factor (IRF), and NF-kB [20].
In future, it will be crucial to further develop more effcient and accurate prognostic tools that incorporate clinical, cytogenetic, and molecular data.Terapeutic strategies and agent design are constantly reformed in order to pursue discoveries about the biology of the disease, the pathways involved, and the mechanisms of drug resistance.
To capture the latest discoveries, this review focuses on the present knowledge about single gene/gene promoter methylation in CLL biology while also recording older data that have been published in past papers.Trough a review of 126 articles indexed in PubMed, we ended up recording data for 133 genes.Ten, we classifed them in the following tables according to their associated biological pathways and the related data on CLL studies.It is reasonable for several genes to be associated with more than one pathway.A characteristic attempt to capture the correlations between most genes and pathways is presented in Figure 1.
In the following tables are presented the genes we identifed in our search, with information on their function, as well as the fndings of studies regarding CLL.In each table, we introduced in detail, data on the study of methylation or mRNA expression of single genes in CLL patients that showed interesting correlations with clinical and laboratory data.1).SMYD3 is a chromatin modifer that is involved in the development and progression of several malignancies.Methylation levels in specifc SMYD3 gene promoter CpG sites were reported to independently predict time to treatment [29].

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Hypermethylation of the hTERT gene in CLL was one of the frst to be described.TERT acts as a subunit of telomerase, preventing chromosomal degradation after DNA replication.It was reported that hTERT promoter hypermethylation led to decreased telomerase activity and was associated with superior overall survival [30].2).Ribonucleotide reductase is required for DNA replication and repair and consists of RRM1 and RRM2 proteins.In CLL patients, RRM1 mRNA expression was higher in patients without anemia, absence of lymphadenopathy, and 17p gene deletion.Moreover, abnormal lactate dehydrogenase (LDH) levels and higher Rai stage were associated with lower RRM1 mRNA levels.Higher expression of RRM2 mRNA was detected in patients without lymphadenopathy, Rai stage 0, and trisomy 12. Te methylated status of RRM1 promoter signifcantly correlated with lymphadenopathy presence.Te methylated status of 4

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Table 1: Chromosome maintenance and cell cycle process.

Related pathways
Findings in CLL Ref.

SMYD3
Chromatin organization Methylation levels in specifc CpG sites independently predicted time to treatment [29] hTERT Chromosome maintenance signaling by WNT Promoter hypermethylation was associated with superior overall survival [30] CDKN2A/ 2B Capable of inducing cell cycle arrest in G1 and G2 phases and regulation of activated PAK-2 gene expression (transcription) Both genes' promoters have been found variously hypermethylated in CLL cases among diferent studies; common point of these studies was that hypermethylation of CDKN2A and CDKN2B was mutually exclusive in CLL cases [31][32][33][34] KLF4 G1-to-S transition of the cell cycle after DNA damage through p53 gene expression (transcription), NOTCH Aberrant methylation of the KLF4 promoter was signifcantly associated with gene expression levels compared to normal samples after B cell activation, KLF4 expression was reported to be downregulated [35] PTPN6 Cell growth, diferentiation, mitotic cycle, and oncogenic transformation In advanced Rai stage cases, aberrant methylation of PTPN6 promoter reached 70% in the samples examined [36] AGBL4 Metabolism of proteins actin and tubulin folding AGBL4 expression was reported to be reduced in patients with hypermethylated promoter regions and hypomethylated body regions [37,38] CLL: chronic lymphocytic leukemia, Ref.: reference, SMYD3: SE translocation and MYN-domain containing 3 protein, hTERT: human telomerase reverse transcriptase, CDKN2A/2B: cyclin-dependent kinase inhibitors 2A and 2B, KLF4: Krüppel-like factor 4, PTPN6: tyrosine-protein phosphatase nonreceptor type 6, AGBL4: ATP/GTP binding protein like 4, WNT: wingless-related integration site, PAK-2: P21 activated kinase 2, NOTCH: neurogenic locus notch homolog protein.transcription factor with an important role in the regulation of hematopoiesis and nervous system development [66].

Integrin Subunit Alpha 4 (ITGA4) Gene (Table 5).
ITGA4 is capable of cell adhesion molecules and fbronectin binding and is involved in apoptotic and integrin pathways.
It is reported to be deregulated in CLL with adverse clinical features, suggested as a negative prognostic factor related to a more aggressive course and shorter time to treatment.Protein expression is regulated at the mRNA level and in a methylation-regulated manner.Hypermethylation at specifc ITGA4 CpG sites was a common phenomenon in the del13q14+ samples.Moreover, it was demonstrated that the methylation status of the ITGA4 gene at CpG site 1 may have a prognostic role [70].

Doublecortin-Like Kinase 2 (DCLK2) and Tumor Necrosis Factor Receptor Superfamily Member 1B (TNFRSF1B)
Genes (Table 6).Retrotransposons (also referred as class I transposable elements or transposons through RNA intermediates) are a type of genetic component that copy and paste themselves into diferent genomic locations (transposons) by retroconverting RNA into DNA via the reverse transcription process using RNA transposition intermediate.In solid neoplasms, universal hypomethylation of these elements has been described.In CLL, locus-specifc hypomethylation was detected with diferential expression of proximal genes, including DCLK2 and TNFRSF1B genes.Moreover, higher levels of DCLK2 and TNFRSF1B expression were associated with inferior survival.DCLK2 protein is characterized by transferase and protein tyrosine kinase activity, whereas TNFRSF1B protein by ubiquitin protein ligase binding and tumor necrosis factor (TNF) receptor activity.Both proteins are involved in TGF-β and TNF pathways [88].
WNT5A gene encodes a receptor tyrosine kinase-like orphan receptor (ROR1) ligand with DNA-binding transcription factor activity and is involved in the regulation of p21 activated kinase 2 (PAK-2), WNT signaling, and developmental pathways during embryogenesis.It is known that its expression difers in CLL patients with worse prognosis in the IGVH-mutated subgroup.Methylation levels of all CpG sites in the WNT5A gene promoter were lower in the group of the intermediate genome methylation profle.In the memory-like and intermediate genome methylation profle groups, promoter methylation and subsequent undetectable WNT5A expression correlated with longer treatment-free survival [95].8).NFATC1 is part of the NFAT transcription complex.It is primarily involved in gene transcription during the immune response and acts as a downstream regulator of the BCR signaling pathway.As with many other proteins, alternative splicing leads to multiple transcription variants, which in turn may induce the expression of diferent cytokine genes.Moreover, NFATC1 protein is a central target for immunosuppressive agents.Regarding CLL, when the genome-wide DNA methylation study was performed, NFATC1 gene was reported to be hypomethylated and upregulated.Moreover, NFATC1 gene promoter DNA hypomethylation correlated inversely with RNA levels and was associated with Binet disease staging and thymidine kinase levels, suggesting a potential central role of NFATC1 in CLL pathobiology [105].9).LDOC1 gene encodes a leucine zipper protein and is suggested to regulate the NF-κB pathway through the plasma membrane ATPase or TNFmediated pathway of apoptosis.Its expression has been studied in oral squamous cell carcinoma and pancreatic cancer cells.Regarding CLL, unmethylated status was found in IGVH-unmutated cases [26,111,112].

Related pathway
Findings in CLL Ref.

CTLA4
Gene expression (transcription) CD28 costimulation CTLA4 was hypomethylated in the frst exon region and body region and had 128-fold higher expression compared to healthy controls [37,59]

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Related pathway
Findings in CLL Ref.

CD5
Hematopoietic stem cells and lineage-specifc markers, TCR signaling CD5 has been reported to be highly expressed in CLL cells and it is demonstrated to be hypomethylated in promoter and body regions [37,94] NFATC1 Regulation of activated PAK-2, activation of cAMP-dependent PKA downstream regulator of the BCR signaling pathway Trough genome-wide DNA methylation, NFATC1 was reported to be globally hypomethylated and, therefore, upregulated NFATC1 promoter hypomethylation correlated inversely with RNA levels and was associated with Binet disease staging and thymidine kinase levels   11).In a small Korean cohort, genome-wide methylation profling identifed the CREBBP gene with no known relevance to CLL to be diferentially methylated among other genes previously known to be afected in CLL.CREBBP gene encodes chromatinmodifying enzymes and has been described in difuse large B cell lymphoma, acute lymphoblastic leukemia, and lung cancer [130].

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2.12.JAK-STAT Pathway 2.12.1.SOCS-1 (Suppressor of Cytokine Signaling 1) Gene (Table 12).SOCS-1 is an inhibitor of cytokine signal transduction.Its gene expression can be positively afected by interleukins 2 and 3, erythropoietin, granulocyte macrophage colony-stimulating factor, and interferon-gamma.Gene polymorphisms of SOCS1 and its expression have been studied in several malignancies including difuse large B-cell lymphoma and acute lymphoblastic leukemia, where, particularly, the expression level of SOCS1 was lower compared to the control group.In CLL, the SOCS1 gene body was hypermethylated in no case [135].

T-Cell Leukemia/Lymphoma 1 Oncogene (TCL1A)
Gene (Table 13).Abnormal expression of TCL1A gene in mouse B-cells led to a leukemia phenotype similar to aggressive human CLL.It is demonstrated that TCL1A physically interacts with DNA methylthansferases 3A and 3B.TCL1A is suggested to act as a coactivator of Akt, activator protein 1 (AP1), and NF-κB pathways with a potential involvement in CLL cells resistance to apoptotic mechanisms.TCL1A expression seems to be strongly associated with the expression levels of ataxia-telangiesctasia mutated (ATM) gene in malignant and nonmalignant B cells.TCL1A gene promoter was hypomethylated in CLL cells and signifcantly correlated with TCL1A transcription enhancement [139].
2.14.Rho GTPases/Rhodopsin-Like Receptors 2.14.1.Endothelin-1 (ET-1) Gene (Table 14).Tis gene is responsible for the creation of a peptide that belongs to the endothelin/sarafotoxin family.Tis peptide acts as a potent vasoconstrictor and its receptors have been well studied as therapeutic targets in the treatment of pulmonary arterial hypertension.Regarding ET-1 gene expression, alternative splicing leads to several transcript variants and abnormal expression is thought to promote tumorigenic activity, with ET-1 expression being under the constant control of the NF-kB signaling pathway.In CLL, ET-1 is reported to be involved in survival, drug resistance, and growth signaling of leukemic cells.Moreover, it is reported that basal expression levels of ET-1 are afected when high methylation in the region of ET-1 gene frst intron is detected [141].

Ubiquitin Conjugating Enzyme E2 R2 (UBE2R2) Gene
(Table 15).UBE2R2 is involved in Class I MHC-mediated pathways as well as in the metabolism of certain proteins.To develop a tool useful in stratifying CLL patients on a specifc methylation signature basis and focusing on time to treatment, it was demonstrated that UBE2R gene methylation levels independently predicted time to treatment [29].16).ESR1 gene encodes a ligand-dependent transcription factor.ESR1 acts through direct binding to specifc estrogen response elements and is associated with other transcription factors.ESR-mediated signaling has been studied in several solid tumors and hematological malignancies.When studied in CLL samples, ESR1 gene was amplifed in 15% of the samples examined with a copy number loss frequency ranging between 1 and 10%.Te ESR1 gene promoter region was methylated in one out of ten CLL samples controlled, and in normal B cell, the gene promoter was completely unmethylated [51].

Related pathway
Findings in CLL Ref.

Related pathway
Findings in CLL Ref.
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Related pathway
Findings in CLL Ref.
TCL1A PI3K-Akt signaling AP1 pathway NF-kB ATM TCL1A promoter was reported to be hypomethylated in CLL cells and correlated with signifcant TCL1A transcription enhancement [139] PHLPP1 PIP3 signaling PI3K-Akt signaling Low PHLPP1 expression is reported parallel with its mRNA levels; further analysis detected that the end region of exon 1 may be important in the regulation of PHLPP1 expression although low methylation was observed in the promoter region; compared with normal B cells, the CLL cells with absent or low PHLPP1 expression displayed signifcantly higher CpG methylation levels and more methylated CpG sites compared than normal B cells and PHLPP1-expressing CLL cells; methylation inhibition led to moderate regulation of PHLPP1 expression [140] CLL: chronic lymphocytic leukemia, Ref.: reference, TCL1A: T-cell leukemia/lymphoma 1 oncogene, PHLPP1: PH domain and leucine rich repeat protein phosphatase 1, PI3K-Akt: phosphatidylinositol-3 kinase-protein kinase B, AP1: activator protein 1, NF-kB: nuclear factor kappa-beta, ATM: ataxia-telangectasia mutated, PIP3: phosphatidylinositol (3,4,5)-trisphosphate.

Related pathway
Findings in CLL Ref.
ET-1 GPCR signaling, class A/1 (Rhodopsin-like receptors), NF-kB ET-1 is involved in survival, drug resistance, and growth signaling of leukemic cells; basal expression levels of ET-1 are afected when high methylation levels in a region of ET-1 frst intron are detected [141] PLD1 Cell survival and protection from apoptosis.Glycerophospholipid biosynthesis signaling by Rho GTPases Unmethylated PLD1 gene body was reported in IGVH-mutated cases [26,142]

DLC1
Signaling by Rho GTPases In advanced Rai stage CLL cases, aberrant methylation of DLC1 promoter was identifed in 89.7% of the samples examined [36] S1PR4 Involved in cell migration, GPCR signaling, class A/1 (Rhodopsin-like receptors) In a study where cells from CLL, Richter's transformed CLL and normal B cells were analyzed, and S1PR4 displayed signifcantly higher promoter methylation levels in Richter's syndrome compared to the other groups [85] GHSR Class A/1 (Rhodopsin-like receptors), GPCR signaling Remarkable hypermethylation at the promoter region and frst exon of the gene was detected; abnormal methylation was able to distinguish with high sensitivity and specifcity malignant from normal cells; GHSR hypermethylation was reported to be identifed even in early disease stages [143] CLL: chronic lymphocytic leukemia, Ref.: reference, PLD1: phospholipase D1, DLC1: deleted in liver cancer 1, S1PR4: sphingosine 1-phosphate receptor 4, ET-1: endothelin 1, GHSR: growth hormone secretagogue receptor type 1, GPCR: G protein-coupled receptor, NF-kB: nuclear factor kappa-beta, IGVH: immunoglobulin variable heavy chain gene. 20

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Table 15: Class I MHC-mediated antigen processing.

Related pathway
Findings in CLL Ref.

VHL Class I MHC-mediated antigen processing and presentation
Methylated promoter status was reported in IGVH-unmutated CLL [14,144] UBE2R2 Class I MHC-mediated antigen metabolism of proteins Methylation levels in specifc CpG sites independently predicted time to treatment with some of them being located in the gene body [29] CLL: chronic lymphocytic leukemia, Ref.: reference, MHC: major histocompatibility complex, VHL: von Hippel-Lindau, UBE2R2: ubiquitin conjugating enzyme E2 R2, IGVH: immunoglobulin variable heavy chain gene.

Related pathway
Findings in CLL Ref.

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Table 19: Drug uptake mechanisms and sensitivity.

Related pathway
Findings in CLL Ref.

SLC22A18
Drug sensitivity cellular metabolism and growth Unmethylated gene body status was reported in IGVH-unmutated samples [26,162] SLCO3A1 Transport of inorganic cations/anions and amino acids/oligopeptides and transport of vitamins, nucleosides, and related molecules, drug uptake mechanisms Diferentially methylated regions of the SLCO3A gene have been associated with survival features in CLL cohorts where the hypermethylated status was linked to inferior post-treatment survival [66] CLL: chronic lymphocytic leukemia, Ref.: reference, SLC22A18: solute carrier family 22, member 18, SLCO3A1: solute carrier organic anion transporter family member 3A1, IGVH: immunoglobulin variable heavy chain gene.

Related pathway
Findings in CLL Ref.

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Table 22: Various other genes were investigated in CLL (cont.).

Related pathway
Findings in CLL Ref.

REPS1
Vesicle-mediated transport and clathrin-mediated endocytosis Methylation levels in specifc CpG sites independently predicted time to treatment with some of them being located in the gene body [29] ATP9B Ion channel transport and cardiac conduction Methylation levels in specifc CpG sites independently predicted time to treatment with some of them being located in the gene body [29] GRB2 Downstream signaling of activated FGFR2 and prolactin signaling Genome-wide methylation profling identifed GRB2 gene body to be diferentially methylated [130] DLEU7 Analysis did not detect any specifc mutations in DLEU7, but DLEU7 expression was absent in CLL cells; methylation of a CpG island in the promoter region of DLEU7 was identifed as a possible explanation for the absence of DLEU7 expression, with the promoter reported to be methylated in most of the CLL samples [179] ADAMTS17 Metabolism of proteins immune cell transmigration, VCAM-1/CD106 signaling, gene expression (transcription).MAGuK tumor suppressor pathway ADAMTS17 expression was reduced in patients with hypermethylated promoter regions and hypomethylated body regions [37,180] KCNG2 Integration of energy metabolism potassium channels KCNG2 expression was reported to be reduced in patients with hypermethylated promoter regions and hypomethylated body regions [37,181] ME3 Respiratory electron transport ATP synthesis TCA cycle ME3 expression was reported to be reduced in patients with hypermethylated promoter regions and hypomethylated body regions [37,182] CLL: chronic lymphocytic leukemia, Ref.: reference, REPS1: RalA-binding protein-associated Eps domain-containing 1, ATP9B: ATPase phospholipid transporting 9B, GRB2: growth factor receptor-bounding protein 2, DLEU7: deleted in lymphocytic leukemia 7, ADAMTS17: a disintegrin and metalloproteinase with thrombospondin motifs 17, KCNG2: potassium voltage-gated channel modifer subfamily G member 2, ME3: malic enzyme 3, FGFR2: fbroblast growth factor receptor 2, VCAM-1: vascular cell adhesion protein 1, CD106: cluster of diferentiation 106, MAGuK: membrane-associated guanylate kinase, ATP: adenosine triphosphate, TCA: tricarboxylic acid.
28 Advances in Hematology  21 and  22).Specifc CpG sites at which the methylation levels independently predicted time to treatment were detected and some of them located in the gene bodies of REPS1

Conclusion
Some studies have focused on the potential clinical impact of DNA methylation in CLL phenotypes.Based on the fndings abovementioned, time to treatment, an essential feature of CLL management, was reported to be predicted by the methylation status of SMYD3 gene promoter and UBE2R gene methylation levels [29].Methylation levels in specifc CpG sites located in the gene bodies of REPS1, IL1B, and ATP9B genes also correlated with the time to treatment [29].
Regarding overall survival, hTERT gene promoter hypermethylation was associated with superior overall survival [30], and specifc hypermethylated MAFB gene loci correlated with inferior post-treatment survival [66].Moreover, locus-specifc hypomethylation of retrotransposons proximal to DCLK2 and TNFRSF1B was related with higher levels of DCLK2 and TNFRSF1B expression and subsequent inferior survival [88].When CLL patients were classifed upon specifc IGVH mutation patterns, promoter methylation of the WNT5A gene showed longer treatment-free survival in a subgroup of the patients [95].Lower levels of DNA methylation of the ANGPT2 gene were related to a particularly poor prognosis in CLL patients [14,121].
Findings that could be useful in staging and correlating methylation with pre-existing prognostic factors in CLL include RRM1/2 expression, the promoter methylation status that correlated with lymphadenopathy, 17p gene deletion, Rai stage, and trisomy 12 [39], and the Advances in Hematology documentation of hypermethylation at specifc ITGA4 CpG sites in del13q14+ samples [70].NFATC1 gene promoter hypomethylation was associated with Binet disease staging [105].
Drug sensitivity and mechanisms of resistance development are another important area of study in CLL.Hypermethylation of specifc SLCO3A1 gene loci, a gene which among others is involved in drug uptake mechanisms, has also been associated with inferior post-treatment survival [66].Another gene, ET-1, is related with drugresistance potential and its expression is afected upon high methylation levels in its frst intron region in patients with CLL [141].
A last fnding that could theoretically have a clinical application in CLL cases is about the SHANK1 gene.A specifc CpG locus of its body (hg19) was hypermethylated when compared to control samples.Also, it was even weakly supported that methylation in the same CpG was detectable in blood samples collected years before CLL diagnosis [170].It is a fnding that, if confrmed, is very interesting and could be used in panels for early diagnosis of the disease, certainly in combination with other markers in the context of a population screening program or the surveillance of highrisk patients.Certainly, such methods of investigation have their peculiarities and must be applied after appropriate research and discussion in order to be necessary or benefcial for the patients.
Our fndings suggested that certain methylation patterns have been associated with disease progression and survival features and may be used as prognostic markers.In addition, some studies have investigated the use of hypomethylating agents as a part of a therapeutic strategy in CLL.
Regarding fndings from previous studies that focused on studying large numbers of genes, data are generally limited but quite intriguing.Kanduri et al. applied highresolution methylation microarrays (27 578 CpG sites) to CLL samples, which were classifed in IGHV-mutated (favorable) and IGHV-unmutated/IGHV3-21 (poorprognostic) subsets.Results demonstrated signifcant differences in methylation patterns between these subgroups.In IGHV-unmutated cases, they reported methylation of known or potent tumor suppressor genes (for example, VHL, ABI3, and IGSF4) as well as unmethylated genes involved in cell growth and tumor progression (ADORA3 and PRF1).In contrast, these latter genes were silenced by methylation in IGHV-mutated patients.Moreover, they reported the reinducing of four methylated tumor suppressor genes (including VHL and ABI3) in IGHVunmutated samples using the methyl inhibitor 5-aza-2′deoxycytidine [26].Pei et al. reported a genome-wide DNA methylation analysis with 1,764 gene promoters being identifed as diferentially methylated in at least one sample when compared with normal B cell samples.Aberrant hypermethylation was discovered in all HOX gene clusters and a signifcant number of WNT signaling pathway genes.Te NFATc1p2 promoter and frst intron hypomethylated status correlated with the upregulation of both NFAT protein expressions [25].Kulis et al. performed a wide analysis of the DNA methylome in normal B cells and CLL samples.Tey identifed widespread hypomethylation targeting mainly the gene body and enhancer regions, suggesting that DNA methylation may be functionally relevant beyond promoter regions.Moreover, they reported that distinct patterns in DNA methylation were recorded between diferent CLL subtypes [18].Cahill et al. used high-resolution 450 K arrays to analyze samples from IGHV-mutated or untreated and IGHV-unmutated or treated patients.Tey identifed 2239 diferentially methylated CpG regions between IGHV-mutated and -unmutated patients, where the majority of the regions were placed outside annotated CpG islands.Known CLL prognostic genes (i.e., LPL, ZAP70, and NOTCH1), epigenetic regulators (HDAC9/4 and DNMT3B), B-cell signaling, and numerous TGF-β and NF-κB/TNF pathway genes were diferentially methylated between the subgroups [14].Barrow et al. reported 490 diferentially methylated regions after exposure to therapy.Among them, 31 were CLL related.Seventeen genes were classifed as diferentially expressed, following treatment in an independent cohort.Methylation of the HOXA4, MAFB, and SLCO3A1 diferentially methylated regions associated with posttreatment patient survival and HOXA4 displayed the strongest association.Reinducing of HOXA4 expression in cell lines and primary CLL cells increased apoptosis following treatment with fudarabine, ibrutinib, and idelalisib [66].Lastly, Zhang et al. identifed 34,797 diferentially methylated positions related to CLL.Most of them were hypomethylated and located in gene body sites.Tey combined these positions with existing DNA methylation and RNA sequencing data and identifed regions associated with 1,130 genes whose expression was signifcantly different in CLL samples [37].
Te limitations of this study include possible evidence selection bias because data from statistically signifcant studies are more likely to be published than those that are not statistically signifcant.Moreover, in some limited reports, it was not stated whether the methylation study included the gene body, the promoter region, or some other related loci.Lastly, some papers did not reported correlations with patients' clinical data such as sampling time, staging, or prognostic and treatment features.
To summarize, there are still many mechanisms that need to be investigated in order to defne the extent of global aberrant DNA methylation in diferent prognostic groups, the fundamental role of DNA methylation in sites other than CpG islands, and the interaction of DNA methylation with other regulatory processes in the pathogenesis of CLL.Moreover, with the prospect that there is and will continue to be increasing data on gene methylation, it should be noted that a large number of fndings will concern passenger DNA methylation events that need to be identifed accordingly.
In conclusion, at some points, the research raises even more questions than answers.Further research is still needed to fully understand the complex interplay between DNA methylation and other epigenetic and genetic alterations in CLL and to develop more efective targeted therapies and prognostic stratifcation tools for this disease.

Table 2 :
DNA repair mechanisms and purine biosynthesis.

Table 3 )
[37,59]4 protein is involved in gene transcription and acts as an immune checkpoint to regulate T-cell function.Mutations in its gene have been associated with insulin-dependent diabetes mellitus, Grave's disease, Hashimoto thyroiditis, celiac disease, systemic lupus erythematosus, and other autoimmune diseases.In CLL cases, CTLA4 gene was hypomethylated in the frst exon region and body region.Compared to healthy controls, CTLA4 gene had a 128-fold higher expression in CLL samples[37,59].
CpG island in the promoter region remained methylated both in normal B cells and CLL cells, while variable methylation levels were recorded in the proximal CpG island only in CLL cells exposure of CLL cells to a demethylating agent led to increased catalase mRNA levels CLL: chronic lymphocytic leukemia, Ref.: reference, ZNF540: human zinc fnger protein 540, LMO2: LIM-only protein 2, RARb2: retinoic acid receptor B2, CAT: catalase, CTLA4: cytotoxic T-lymphocyte associated protein 4, MAPK: mitogen-activated protein kinase, IGVH: immunoglobulin variable heavy chain gene.8 Advances in Hematology

Table 4 :
RNA polymerase I promoter opening.

Table 5 :
Programmed cell death/cell apoptosis and p53 signaling.

Table 20 :
Various other genes investigated in CLL.

Table 21 :
Various other genes were investigated in CLL (cont.).

Table 23 )
[183]expression of the CRNDE gene is increased in proliferating tissues and is involved in the expression of genes associated with metabolism and in neoplasms such as colorectal adenomas and adenocarcinomas.Its related transcription is inversely regulated by insulin and insulin-like growth factors.When studied in CLL cell lines, CRNDE was downregulated and the methylation level of CRNDE promoter was higher than in normal B cells.After exposure to demethylating agents, an increase of CRNDE expression levels was reported[183].